CA2341639A1 - Superoxide dismutase as a vaccine antigen - Google Patents
Superoxide dismutase as a vaccine antigen Download PDFInfo
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- CA2341639A1 CA2341639A1 CA002341639A CA2341639A CA2341639A1 CA 2341639 A1 CA2341639 A1 CA 2341639A1 CA 002341639 A CA002341639 A CA 002341639A CA 2341639 A CA2341639 A CA 2341639A CA 2341639 A1 CA2341639 A1 CA 2341639A1
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0089—Oxidoreductases (1.) acting on superoxide as acceptor (1.15)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/10—Antimycotics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
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- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Veterinary Medicine (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Chemical & Material Sciences (AREA)
- Pharmacology & Pharmacy (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Animal Behavior & Ethology (AREA)
- Oncology (AREA)
- Public Health (AREA)
- Communicable Diseases (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
Compositions comprising Cu,Zn-Superoxide dismutase (Cu,Zn-SOD), nucleic acid encoding a Cu,Zn-SOD and/or antibody to a Cu,Zn-SOD are described as well as their use as vaccines. Also described are methods for isolation of Cu,Zn-SOD s and for preparation of pharmaceutical compositions, preferably for providing or eliciting protective immunity to meningococcal infection in an animal.</S DOAB>
Description
SUPEROXIDE DISMUTASE AS A VACCINE ANTIGEN
The present invention relates to pharmaceutical compositions for treating and/or vaccinating against bacterial infection and to methods of manufacturing such compositions. In particular, the invention relates to pharmaceutical compositions which comprise superoxide dismutase or antibodies thereto.
At present most bacterial infections in humans or animals are treated after infection has set in by administration of antibiotic drugs. As many more strains of pathogenic bacteria become resistant to current antibiotics, the range of options open for treatment decreases. Moreover, many antibiotics can cause dangerous side effects upon individuals or animals taking them, for example allergy to penicillin or the toxicity of sulpha drugs. Furthermore, antibiotic treatment is sometimes only effective if the drug is taken regularly over a period of time thus maintaining a constant level of therapeutic agent in circulation. If individuals forget or are unable to maintain the course of antibiotic treatment then it may be rendered ineffective.
. Some bacterial infections progress very quickly, sometimes too quickly for antibiotic treatment to have much effect unless administered at a very early stage in the course of the infection. Meningococcal disease is one example of such a virulent infection and is caused by the pathogen Neisseria meningitides. In many cases symptoms of disease at first resemble those of influenza and thus infected individuals often delay in seeking medical attention. Vaccines based on polysaccharides present on the surface of some N. meningiiidis serogroups are available at present but they show limited protection against infection. Moreover, the surface polysaccharide of serogroup B strains of N, meningitides (causative agent of over half the cases of meningococcal disease in the UK) are only weakly immunogenic and are not included in current vaccines.
The present invention relates to pharmaceutical compositions for treating and/or vaccinating against bacterial infection and to methods of manufacturing such compositions. In particular, the invention relates to pharmaceutical compositions which comprise superoxide dismutase or antibodies thereto.
At present most bacterial infections in humans or animals are treated after infection has set in by administration of antibiotic drugs. As many more strains of pathogenic bacteria become resistant to current antibiotics, the range of options open for treatment decreases. Moreover, many antibiotics can cause dangerous side effects upon individuals or animals taking them, for example allergy to penicillin or the toxicity of sulpha drugs. Furthermore, antibiotic treatment is sometimes only effective if the drug is taken regularly over a period of time thus maintaining a constant level of therapeutic agent in circulation. If individuals forget or are unable to maintain the course of antibiotic treatment then it may be rendered ineffective.
. Some bacterial infections progress very quickly, sometimes too quickly for antibiotic treatment to have much effect unless administered at a very early stage in the course of the infection. Meningococcal disease is one example of such a virulent infection and is caused by the pathogen Neisseria meningitides. In many cases symptoms of disease at first resemble those of influenza and thus infected individuals often delay in seeking medical attention. Vaccines based on polysaccharides present on the surface of some N. meningiiidis serogroups are available at present but they show limited protection against infection. Moreover, the surface polysaccharide of serogroup B strains of N, meningitides (causative agent of over half the cases of meningococcal disease in the UK) are only weakly immunogenic and are not included in current vaccines.
Accordingly, it is an object of the invention to provide a pharmaceutical composition comprising a vaccine antigen or an antibody that effectively protects against or ameliorates bacterial infection. It is a further object of the invention to provide a pharmaceutical composition comprising a vaccine antigen that protects against meningococcal disease. It is yet a further object to provide a vaccine antigen that also provides protective immunity against a broader range of infectious bacteria. It is a further object to provide a method of manufacturing antibodies that can provide protective immunity to a range of bacterial pathogens when included in a pharmaceutical preparation. It is still a further object of the invention to provide a multivalent vaccine which provides protective immunity to a wide range of bacterial infections.
Cu,Zn-Superoxide Dismutase (Cu,Zn-SOD) is an metalloenzyme found in many prokaryotic and eukaryotic organisms. It catalyses the reduction of the superoxide radical anion, 02', to hydrogen peroxide and molecular oxygen, thus playing an important role in the removal of cytotoxic free radicals from the organism. In bacteria Cu,Zn-SODs have been identified in the periplasm of a number of Gram negative species including N. meningitidis, Haemophiius ducreyi, Haemophilus parainfiuenzae, Actinobacillus pieuropneumoniae and Pasteurella multocida (Knoll et al, 1995). The enzyme can exist as a dimer or a monomer, and examples of monomeric Cu,Zn-SODS are those from Brucella abortus and Escherichia coli (Pesce, et al, 1997). It is believed that Cu,Zn-SOD provides a defence for the bacterium against the burst of oxygen free radicals released by phagocytic host cells, such as macrophages, during infection (Wilks et al, 1998; Farrant et al, 1997).
A known attempt to utilise Cu,Zn-SOD as a vaccine antigen has not been successful. Tabatabai (Tabatabai and Pugh, 1994) showed that a synthetic fragment of a monomeric Cu,Zn-SOD (denoted as peptide 3) from B. abortus was able to provide a low level of immunity in mice against Brucella infection, but the level of protection provided was lower than that seen when using Bruceila cell surface proteins and (ipopolysaccharide antigens. Moreover, vaccination with the entire Cu,Zn-SOD failed to provide protective immunity at all. Tabatabai concluded that the antigenic fragment contained within Brucelia Cu,Zn-SOD was counteracted by other parts of the protein which prevented it from eliciting an antigenic activity. This masking property was in fact so strong that even a mixture of synthetic peptides that included peptide 3 elicited no protective immunity to Brucella infection. Thus the study by Tabatabai et al teaches against the use of either fragments of Cu,Zn-SOD
or full length Cu,Zn-SOD as an effective antigen that could provide protective immunity to bacterial infection.
Cu,Zn-SODs from eukaryotes and most Gram negative bacteria form dimers in their native form. However, the Cu,Zn-SODS of E.coii and B. abortus are atypical in that they are normally monomeric (Pesce et al, 1997). In all Gram negative bacteria that produce Cu,Zn-SOD the protein is localised to the periplasmic space between the outer cell wall and the cell membrane.
The present invention relates to the surprising discovery that a monoclonal antibody raised against a recombinant Cu,Zn-SOD fusion protein from A.
pleuropneumoniae has bactericidal activity against N. meningitidis and protects mice against challenge with N. meningitidis. Furthermore, a recombinant, dimeric Cu,Zn-SOD and immunogenic fragments from A.
pleuropneumoniae (a pig pathogen) act as antigen that can confer protective immunity not only against this organism but also against other Gram negative human pathogens such as N. meningitidis, H. ducreyi, H. parainfluenzae and P. multocida. Thus the Cu,Zn-SOD is suitable as a vaccine component against both animal and human bacterial diseases such as meningococcal disease (caused by N, meningitidis) or chancroid (caused by H. ducreyil , or porcine pleuropneumonia (caused by A. pleuropneumoniae).
Accordingly, a first aspect of the present invention provides a composition comprising a Cu,Zn-SOD of the dimeric type, or a fragment, variant or derivative thereof, and a pharmaceutically acceptable carrier. The first aspect of the invention also provides a composition comprising a nucleic acid encoding a Cu,Zn-SOD of the dimeric type, or a fragment, variant or derivative thereof, and a pharmaceutically acceptable carrier. By "dimeric" we mean a SOD that naturally forms dimers under normal conditions, e.g. is found in dimeric form in nature. A pharmaceutically acceptable carrier could comprise an approved adjuvant such as alum or any other adjuvant approved for pharmaceutical purposes. The Cu,Zn-SOD can be from any bacteria, though especially from known pathogenic bacteria, and from N. meningitidis as an example.
The present invention is not to be restricted to the use of full length or wild type Cu,Zn-SOD of the dimeric type. An antigenic fragment of the Cu,Zn-SOD may also be used in a vaccine formulation. The fragment preferably comprises a region of the Cu,Zn-SOD that is on the surface of the protein, although any fragment that confers protective immunity to bacterial infection is suitable; and the term "fragment" is intended to encompass any fragment against which an antibody may be raised which antibody binds intact, full length SOD. Moreover, mutant variants which have been modified to increase antigenicity or fusion protein derivatives between all or a part of a Cu,Zn-SOD
and another protein for the purposes of purification or increasing antigenicity may also be suitable for use in pharmaceutical compositions. Vaccine components of the invention also include derivatives and variants of Cu,Zn-SOD. The term "derivative" is intended to encompass combinations of Cu,Zn-SOD with other proteins or molecules, including carbohydrates to form conjugate vaccines, the derivative retaining antigenicity such that an antibody raised against the derivative binds intact, full length SOD. The term "variant"
is intended to encompass a polypeptide having an amino acid sequence that varies from that of intact, full length SOD, but such that antibodies raised against the variant bind intact, full length SOD.
In a preferred embodiment of the invention the pharmaceutical composition provides protection against meningococcal infection and/or disease. In further preferred embodiments of the invention the pharmaceutical composition provides protective immunity to infection from Actinobacillus species (e.g. A.
pleuropneumoniae, A. actinomycetemcomitans), Pasteurellaceae species (e.g.
P. multocida), Neisseria species (e.g. N. meningitidis), Haemophilus species (e.g. H. influenzae, H. parainfluenzae, H, ducreyi), Escherichia coli, Salmonella species and other bacteria producing a Cu,Zn-SOD. In a specific embodiment of the invention the Cu,Zn-SOD is expressed from a recombinant gene cloned from Actinobacillus pleuropneumoniae.
It is an advantage of the present invention that a Cu,Zn-SOD of the dimeric type, or a fragment, variant or derivative thereof, can confer protective immunity to infection from a broad range of bacterial pathogens. It is of further advantage that the present inventian provides for pharmaceutical compositions comprising Cu,Zn-SODs, or a fragment, variant or derivative thereof, that are protective against bacterial infection in both humans and animals and in particular to meningococcal disease. It is of still further advantage that an antibody to a Cu,Zn-SOD from one species of bacteria can provide protective immunity to infection from a plurality of other species of bacteria, in particular that the invention provides protective immunity to meningococcal disease. A further advantage of the present invention is that Cu,Zn-SOD is relatively abundant and can be easily purified from bacterial cultures. Moreover, recombinant Cu,Zn-SODS can be fused to other proteins, such as glutathione-S-transferases, to facilitate purification from bacterial cultures expressing the fusion protein, and the Cu,Zn-SOD moiety retains both antigenicity and biological activity.
In use of the invention, a Cu,Zn-SOD is cloned from the pig pathogen Actinobacillus pleuropneumoniae, to give a recombinant form of the gene.
The recombinant Cu,Zn-SOD gene is optionally linked to, such as by fusing, a glutathione-S-transferase gene to enable easy purification of the fusion protein when expressed in bacteria. A pharmaceutical preparation is prepared comprising the purified Cu,Zn-SOD protein and a pharmaceutically acceptable carrier; in one use the carrier includes the adjuvant alum. The pharmaceutical composition is suitably administered to the individual via any route. The nature or form of the composition may be selected from any conventional pharmaceutical composition including but not limited to tablets, capsules, oral compositions, liquids, compositions for infusion, syrups, solutions, powdered formulations and granular formulations. The pharmaceutical composition, in use, stimulates the individual to produce antibodies against the antigenic Cu,Zn-SOD protein, some of which provide protective immunity to a broad range of pathogens. In particular the pharmaceutical composition provides protective immunity to meningococcal disease.
In a specific example of the invention in use, described in more detail below, a Cu,Zn-SOD gene - the sodC gene - is isolated from N. meningitidis genomic DNA by standard PCR techniques. The product of the PCR reaction additionally incorporates a His-tag sequence (coding for six histidines at the C terminus of the protein). The isolated sodC gene plus His-tag is cloned into an expression vector and then transformed into E. coli, where the protein product is expressed. The expressed protein is purified on a nickel charged affinity column, to which the His-tag preferentially binds. The SodC protein is then eluted from the affinity column and is suitably incorporated in the pharmaceutical composition of the invention as described above.
A second aspect of the invention provides a vaccine comprising a Cu,Zn-SOD, or a nucleic acid encoding a Cu,Zn-SOD, of the dimeric type, or a fragment, variant or derivative thereof.
In a specific embodiment of the invention the Cu,Zn-SOD of the dimeric type, or a fragment, variant or derivative thereof, is from a recombinant gene cloned from Actinobaciiius pleuropneumoniae, though the invention also encompasses use of native proteins. In a further preferred embodiment the vaccine provides protective immunity against meningococcal meningitis.
Compositions and vaccines of the invention comprising a nucleic acid which encodes a Cu,Zn-SOD or fragment or derivative thereof are suitably prepared comprising the coding sequence inside a microparticle according to the methods of WO-A-97/17063, incorporated herein by reference.
A third aspect of the invention provides a method of preparing a pharmaceutical composition that consists of:-1) cloning a gene for a Cu,Zn-SOD of the dimeric type to obtain a recombinant form of the gene; and 2) (a? synthesising Cu,Zn-SOD from the recombinant gene; and combining said Cu,Zn-SOD with a pharmaceutically acceptable carrier, or (b) combining said gene with a pharmaceutically acceptable carrier.
A fourth aspect of the invention provides for a composition, especially a pharmaceutical preparation comprising an antibody to a Cu,Zn-SOD of the dimeric type, or a fragment, derivative or variant thereof, and a pharmaceutically acceptable carrier. It is optional, but not essential, that the antibody is a monoclonal antibody.
The present invention thus also provides for an antibody preparation that is raised against a dimeric Cu,Zn-SOD, or a fragment, derivative or variant thereof, from one species of Gram negative bacteria and that confers protective immunity to infection from this bacterium and also to infection from a plurality of other Gram negative bacteria. The antibody can be used in pharmaceutical preparations that confer passive immunity to bacterial infection upon a host organism. In the example described in more detail below a monoclonal antibody raised against Cu,Zn-SOD from A.
pleuropneumoniae provides protection against N, meningitides infection. Thus _g-the monoclonal antibody is suitable for use in treating acute cases of meningococcal disease as well as in providing passive immunity to future meningococcal disease.
In a specific embodiment of the invention the antibody provides protective immunity to meningococcal disease. Thus it is suitable for use in preparations that provide passive immunity to infection and also for treatment of individuals already suffering from meningococcal infection.
In further specific embodiments of the invention the antibody provides protective immunity to bacterial infection from a range of bacterial pathogens including Actinobacillus pleuropneumoniae, Pasteurellaceae species, Neisseria species and Haemophilus species.
In a further preferred embodiment of the invention the antibody displays bactericidal activity. This activity may be directly attributed to the antibody itself or mediated via the complement system.
A fifth aspect of the invention provides for a multivalent vaccine comprising a plurality of Cu,Zn-SODs of the dimeric type, or fragments, derivatives or variants thereof, wherein said plurality of Cu,Zn-SODs are from the same or different species of Gram negative bacteria. A specific embodiment of the invention provides a multivalent vaccine comprising a plurality of Cu,Zn-SODs of the dimeric type, or fragments, derivatives or variants thereof, and one or a number of different bacterial proteins, or a fragment, derivative or variant thereof, that is not or are not a Cu,Zn-SOD. A further preferred embodiment of the invention is a multivalent vaccine that provides protective immunity to meningococcal infection.
A sixth aspect of the invention provides for use of a Cu,Zn-superoxide dismutase of the dimeric type, or a fragment, derivative or variant thereof, in the manufacture of a medicament, e.g. a vaccine, for use against bacterial infection. In a preferred embodiment of the invention the use is in response to a bacterial infection due to Gram negative species of bacteria. In a specific embodiment of the invention the use is in response to a meningococcal infection. This aspect also provides a method of treating bacterial, particularly meningococcal, infection by administering an effective amount of a Cu,Zn-SOD or a fragment, variant or derivative thereof, or other composition according to the invention.
A seventh aspect of the invention provides for an antibody specific to bacterial Cu,Zn-SOD of the dimeric type, or a fragment, derivative or variant thereof. A preferred embodiment of the invention provides for a monoclonal antibody (MAb) that is specific to bacterial Cu,Zn-SOD.
There now follow examples of specific embodiments of the invention.
Example 1 Cu Zn-SOD Monoclonal Antibody Monoclonal antibodies (MAbs) were raised in mice against a glutathione-S
transferase fusion protein with the Cu,Zn-SOD from Actinobacillus pleuropneumoniae. The whole fusion protein as expressed in E. coli was found to be enzymically active. Of 72 potential hybridomas screened by western blotting, two recognised only A, pleuropneumoniae Cu,Zn-SOD, but one also recognised Cu,Zn-SOD from H. parainfluenzae, H. ducreyi, N.
meningitides and A, actinomycetemcomitans. The latter MAb was used for the passive protection studies.
Passive Protection Three experiments were performed and all demonstrated that the MAb protects mice against lethal infections with N, meningitides. Adult NIH mice were given an intra-peritoneal tip) injection with antibody (50,u1) per mouse 2h before challenge with Neisseria meningitides. Mice were given further ip injections of antibody 2, 5 and 24h after the challenge. The N. meningitidis challenge dose was the stated number of viable bacteria (see tables of results), given as an 0.5m1 ip injection, containing 2mg iron dextran. At 24h mice were given a further 2mg iron dextran in an 0.2m1 ip dose. Mice were then observed for 72h and the health of the animals noted. The results of these experiments are shown in the following tables.
WO 00/i2718 PCT/GB99/02828 EXPERIMENT
VACCINE CHALLENGE SURVIVORS
/ CHALLENGED
DOSE
growth 105 5/5 4/5 4/5 medium growth 106 5/5 3l5 3/5 medium 50,u1 MAb 105 515 5/5 5/5 50,u1 MAb 106 5/5 5/5 5/5 5~r1 MAb 105 5/5 5/5 5/5 5NI MAb 106 5/5 4/5 4/5 Polyclonal 105 5/5 5/5 5/5 serum Polyclonal 106 5/5 5/5 5/5 1~ serum '"
- raised against meningococcal outer membrane vesicles.
EXPERIMENT
VACCINE CHALLENGE SURVIVORSJCHALLENGED
Growth 2 x 105 5/5 5l5 5!5 medium Growth 2 x 1 Os 515 1 /5 0/5 medium 50NI MAb 2 x 105 5!5 5/5 5/5 IO 50,u1 MAb 2 x 108 5/5 5/5 5/5 5,u1 MAb 2 x 105 5/5 5/5 5/5 5N1 MAb 2 x 106 5l5 3/5 3/5 Polyclonal 2 x 105 5/5 5/5 5l5 serum *' Polyclonal 2 x 106 5/5 5l5 5/5 serum ~' * - raised against meningococcal outer membrane vesicles.
WO 00/12718 PCTlGB99/02828 EXPERIMENT
VACCINE CHALLENGE SURVIVORS
/ CHALLENGED
Growth 1 x 10' 5/5 0/5 0/5 0/5 medium 50,u1 MAb 1 x 10' 5/5 4/5 3/5 2/5 5,u1 MAb 1 x 10' 5/5 1 /5 0/5 0/5 Polyclonal 1 x 10' 5/5 1 /5 0/5 0/5 serum 50N1 Yersinia1 x 10' 5/5 1 /5 0/5 0/5 MAb 5irl Yersinia1 x 10' 5/5 0/5 0/5 0/5 MAb Bactericidal Activity In addition to passive protective activity, the Cu,Zn-SOD MAb has been found to be bactericidal to two strains of meningococcus, with titres of 64-128 compared to a titre of 256 for a Group B polysaccharide specific MAb.
Example 2 Cloning and Expression of Cu,Zn-SOD - Purification of protein via a nickel affinity column Genomic DNA isolated from N.meningitidis strain MC58 was used as a template to amplify the sodCgene (Cu,Zn-SOD). Primers were designed using the information in the Genbank database. The 5' primer fSEQ ID N0.1 ) incorporates an Ndel site at the level of the ATG start codon, allowing the PCR product to be cloned into a variety of pET (Novagen) and pMTL (CAMR) vectors for expression. The 3' primer (SEQ ID N0.2) generates a polyhistidine (6 x His) tag in the translated protein to facilitate nickel affinity purification.
The primer sequences are as follows:
"SEQ ID N0.1 " : 5' primer: 5' GGC ATA TGA ATA TGA AAA CCT TAT TAG
3' "SEQ ID N0.2" : 3' primer: 5' GGG CTG AGC TTA TTA GTG GTG GTG GTG
GTG GTG TTT AAT CAC GCC ACA TGC CAT ACG TG 3' The products were sub-cloned into pET22b and transferred to BL21 DE3 for initial expression. His-tagged protein was purified on a nickel charged Hi-Trap column.
Example 3 Purification of protein via a glutathione affinity column - A glutathione-S-transferase (GST) fusion was generated by cloning the sodC
gene of Example 2 (described above) into one of the pGEX series of vectors (Pharmacia). An N-terminal fusion was generated by cloning a sodC PCR
product as an EcoRl-Xhol fragment in frame into pGEX-4T-1 (restriction sites generated by PCR primers containing these sites in-frame immediately upstream or downstream of the sodC coding sequence, including 3' stop codon). The expressed protein was a GST-SodC fusion linked by a thrombin cleavage site. The recombinant protein was purified on a glutathione affinity column and cleaved with thrombin to release SodC. This SodC protein has an additional five amino acids on the N terminus, compared to wild type protein (sequence GSPQF).
Example 4 Purification of protein via a glutathione affinity column - Alternative method This alternative cloning strategy varies from the method of Example 3 in that it allows reduction in the number of additional N terminal amino acids on the cleaved SodC product. This is achieved by removing the internal MC58 sodC
BamHl restriction site by site directed mutagenesis. PCR amplification of this altered gene with a 5' BamHl site in-frame upstream of the ATG start codon facilitates cloning of a BamHl-EcoRl fragment into pGEX-2T vector (Pharmacia). The resultant GST-SodC fusion protein is expressed and SodC
released by cleavage with thrombin protease, giving rise to SodC product with just a two amino acid N terminal 'tag' (sequence GS).
References:
Farrant, J.L. et al. (1997); Bacterial copper- and zinc- cofactored superoxide dismutase contributes to the pathogenesis of systemic saimonellosis;
s Molecular Microbiology 25(4), 785-796.
Kroll, J.S., Langford, P.R, Wilks, K.E., Keil, A.D. (1995); Bacterial Cu,Zn-superoxide dismutase: phylogenetically distinct from the eukaryotic enzyme, and not so rare after all!; Microbiology 141, 2271-2279.
io Pesce, A. et al (1997); Unique structural features of the monomeric Cu,Zn-superoxide dismutase from Escherichia coli revealed by X-ray crystallography;
J. Mol. Biol 274, 408-420.
is Tabatabai, L.B., Pugh, G.W. (1994); Modulation of immune responses in Balb/c mice vaccinated with Brucella abortus Cu,Zn-superoxide dismutase synthetic peptide vaccine; Vaccine 12( 10), 919-924.
Wilks, K.E. et al. (1998); Periplasmic superoxide dismutase in meningococcal 2o pathogenicity; Infection and Immunity, 66., 213-217.
SEQUENCE LISTING
<110> Microbiological Research Authority Imperial College School of Medicine Gorringe, Andrew Richard .
Langford, Paul Richard Kroll, John Simon Robinson, Andrew <120> SUPEROXIDE DISMUTASE AS A VACCINE ANTIGEN
<130> 20860pct camr <140>
<141>
<160> 2 <170> PatentIn Ver. 2.1 <210> 1 <211> 27 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: primer <400> 1 ggcatatgaa tatgaaaacc ttattag 27 <210> 2 <211> 59 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: primer <400> 2 gggctgagct tattagtggt ggtggtggtg gtgtttaatc acgccacatg ccatacgtg 59
Cu,Zn-Superoxide Dismutase (Cu,Zn-SOD) is an metalloenzyme found in many prokaryotic and eukaryotic organisms. It catalyses the reduction of the superoxide radical anion, 02', to hydrogen peroxide and molecular oxygen, thus playing an important role in the removal of cytotoxic free radicals from the organism. In bacteria Cu,Zn-SODs have been identified in the periplasm of a number of Gram negative species including N. meningitidis, Haemophiius ducreyi, Haemophilus parainfiuenzae, Actinobacillus pieuropneumoniae and Pasteurella multocida (Knoll et al, 1995). The enzyme can exist as a dimer or a monomer, and examples of monomeric Cu,Zn-SODS are those from Brucella abortus and Escherichia coli (Pesce, et al, 1997). It is believed that Cu,Zn-SOD provides a defence for the bacterium against the burst of oxygen free radicals released by phagocytic host cells, such as macrophages, during infection (Wilks et al, 1998; Farrant et al, 1997).
A known attempt to utilise Cu,Zn-SOD as a vaccine antigen has not been successful. Tabatabai (Tabatabai and Pugh, 1994) showed that a synthetic fragment of a monomeric Cu,Zn-SOD (denoted as peptide 3) from B. abortus was able to provide a low level of immunity in mice against Brucella infection, but the level of protection provided was lower than that seen when using Bruceila cell surface proteins and (ipopolysaccharide antigens. Moreover, vaccination with the entire Cu,Zn-SOD failed to provide protective immunity at all. Tabatabai concluded that the antigenic fragment contained within Brucelia Cu,Zn-SOD was counteracted by other parts of the protein which prevented it from eliciting an antigenic activity. This masking property was in fact so strong that even a mixture of synthetic peptides that included peptide 3 elicited no protective immunity to Brucella infection. Thus the study by Tabatabai et al teaches against the use of either fragments of Cu,Zn-SOD
or full length Cu,Zn-SOD as an effective antigen that could provide protective immunity to bacterial infection.
Cu,Zn-SODs from eukaryotes and most Gram negative bacteria form dimers in their native form. However, the Cu,Zn-SODS of E.coii and B. abortus are atypical in that they are normally monomeric (Pesce et al, 1997). In all Gram negative bacteria that produce Cu,Zn-SOD the protein is localised to the periplasmic space between the outer cell wall and the cell membrane.
The present invention relates to the surprising discovery that a monoclonal antibody raised against a recombinant Cu,Zn-SOD fusion protein from A.
pleuropneumoniae has bactericidal activity against N. meningitidis and protects mice against challenge with N. meningitidis. Furthermore, a recombinant, dimeric Cu,Zn-SOD and immunogenic fragments from A.
pleuropneumoniae (a pig pathogen) act as antigen that can confer protective immunity not only against this organism but also against other Gram negative human pathogens such as N. meningitidis, H. ducreyi, H. parainfluenzae and P. multocida. Thus the Cu,Zn-SOD is suitable as a vaccine component against both animal and human bacterial diseases such as meningococcal disease (caused by N, meningitidis) or chancroid (caused by H. ducreyil , or porcine pleuropneumonia (caused by A. pleuropneumoniae).
Accordingly, a first aspect of the present invention provides a composition comprising a Cu,Zn-SOD of the dimeric type, or a fragment, variant or derivative thereof, and a pharmaceutically acceptable carrier. The first aspect of the invention also provides a composition comprising a nucleic acid encoding a Cu,Zn-SOD of the dimeric type, or a fragment, variant or derivative thereof, and a pharmaceutically acceptable carrier. By "dimeric" we mean a SOD that naturally forms dimers under normal conditions, e.g. is found in dimeric form in nature. A pharmaceutically acceptable carrier could comprise an approved adjuvant such as alum or any other adjuvant approved for pharmaceutical purposes. The Cu,Zn-SOD can be from any bacteria, though especially from known pathogenic bacteria, and from N. meningitidis as an example.
The present invention is not to be restricted to the use of full length or wild type Cu,Zn-SOD of the dimeric type. An antigenic fragment of the Cu,Zn-SOD may also be used in a vaccine formulation. The fragment preferably comprises a region of the Cu,Zn-SOD that is on the surface of the protein, although any fragment that confers protective immunity to bacterial infection is suitable; and the term "fragment" is intended to encompass any fragment against which an antibody may be raised which antibody binds intact, full length SOD. Moreover, mutant variants which have been modified to increase antigenicity or fusion protein derivatives between all or a part of a Cu,Zn-SOD
and another protein for the purposes of purification or increasing antigenicity may also be suitable for use in pharmaceutical compositions. Vaccine components of the invention also include derivatives and variants of Cu,Zn-SOD. The term "derivative" is intended to encompass combinations of Cu,Zn-SOD with other proteins or molecules, including carbohydrates to form conjugate vaccines, the derivative retaining antigenicity such that an antibody raised against the derivative binds intact, full length SOD. The term "variant"
is intended to encompass a polypeptide having an amino acid sequence that varies from that of intact, full length SOD, but such that antibodies raised against the variant bind intact, full length SOD.
In a preferred embodiment of the invention the pharmaceutical composition provides protection against meningococcal infection and/or disease. In further preferred embodiments of the invention the pharmaceutical composition provides protective immunity to infection from Actinobacillus species (e.g. A.
pleuropneumoniae, A. actinomycetemcomitans), Pasteurellaceae species (e.g.
P. multocida), Neisseria species (e.g. N. meningitidis), Haemophilus species (e.g. H. influenzae, H. parainfluenzae, H, ducreyi), Escherichia coli, Salmonella species and other bacteria producing a Cu,Zn-SOD. In a specific embodiment of the invention the Cu,Zn-SOD is expressed from a recombinant gene cloned from Actinobacillus pleuropneumoniae.
It is an advantage of the present invention that a Cu,Zn-SOD of the dimeric type, or a fragment, variant or derivative thereof, can confer protective immunity to infection from a broad range of bacterial pathogens. It is of further advantage that the present inventian provides for pharmaceutical compositions comprising Cu,Zn-SODs, or a fragment, variant or derivative thereof, that are protective against bacterial infection in both humans and animals and in particular to meningococcal disease. It is of still further advantage that an antibody to a Cu,Zn-SOD from one species of bacteria can provide protective immunity to infection from a plurality of other species of bacteria, in particular that the invention provides protective immunity to meningococcal disease. A further advantage of the present invention is that Cu,Zn-SOD is relatively abundant and can be easily purified from bacterial cultures. Moreover, recombinant Cu,Zn-SODS can be fused to other proteins, such as glutathione-S-transferases, to facilitate purification from bacterial cultures expressing the fusion protein, and the Cu,Zn-SOD moiety retains both antigenicity and biological activity.
In use of the invention, a Cu,Zn-SOD is cloned from the pig pathogen Actinobacillus pleuropneumoniae, to give a recombinant form of the gene.
The recombinant Cu,Zn-SOD gene is optionally linked to, such as by fusing, a glutathione-S-transferase gene to enable easy purification of the fusion protein when expressed in bacteria. A pharmaceutical preparation is prepared comprising the purified Cu,Zn-SOD protein and a pharmaceutically acceptable carrier; in one use the carrier includes the adjuvant alum. The pharmaceutical composition is suitably administered to the individual via any route. The nature or form of the composition may be selected from any conventional pharmaceutical composition including but not limited to tablets, capsules, oral compositions, liquids, compositions for infusion, syrups, solutions, powdered formulations and granular formulations. The pharmaceutical composition, in use, stimulates the individual to produce antibodies against the antigenic Cu,Zn-SOD protein, some of which provide protective immunity to a broad range of pathogens. In particular the pharmaceutical composition provides protective immunity to meningococcal disease.
In a specific example of the invention in use, described in more detail below, a Cu,Zn-SOD gene - the sodC gene - is isolated from N. meningitidis genomic DNA by standard PCR techniques. The product of the PCR reaction additionally incorporates a His-tag sequence (coding for six histidines at the C terminus of the protein). The isolated sodC gene plus His-tag is cloned into an expression vector and then transformed into E. coli, where the protein product is expressed. The expressed protein is purified on a nickel charged affinity column, to which the His-tag preferentially binds. The SodC protein is then eluted from the affinity column and is suitably incorporated in the pharmaceutical composition of the invention as described above.
A second aspect of the invention provides a vaccine comprising a Cu,Zn-SOD, or a nucleic acid encoding a Cu,Zn-SOD, of the dimeric type, or a fragment, variant or derivative thereof.
In a specific embodiment of the invention the Cu,Zn-SOD of the dimeric type, or a fragment, variant or derivative thereof, is from a recombinant gene cloned from Actinobaciiius pleuropneumoniae, though the invention also encompasses use of native proteins. In a further preferred embodiment the vaccine provides protective immunity against meningococcal meningitis.
Compositions and vaccines of the invention comprising a nucleic acid which encodes a Cu,Zn-SOD or fragment or derivative thereof are suitably prepared comprising the coding sequence inside a microparticle according to the methods of WO-A-97/17063, incorporated herein by reference.
A third aspect of the invention provides a method of preparing a pharmaceutical composition that consists of:-1) cloning a gene for a Cu,Zn-SOD of the dimeric type to obtain a recombinant form of the gene; and 2) (a? synthesising Cu,Zn-SOD from the recombinant gene; and combining said Cu,Zn-SOD with a pharmaceutically acceptable carrier, or (b) combining said gene with a pharmaceutically acceptable carrier.
A fourth aspect of the invention provides for a composition, especially a pharmaceutical preparation comprising an antibody to a Cu,Zn-SOD of the dimeric type, or a fragment, derivative or variant thereof, and a pharmaceutically acceptable carrier. It is optional, but not essential, that the antibody is a monoclonal antibody.
The present invention thus also provides for an antibody preparation that is raised against a dimeric Cu,Zn-SOD, or a fragment, derivative or variant thereof, from one species of Gram negative bacteria and that confers protective immunity to infection from this bacterium and also to infection from a plurality of other Gram negative bacteria. The antibody can be used in pharmaceutical preparations that confer passive immunity to bacterial infection upon a host organism. In the example described in more detail below a monoclonal antibody raised against Cu,Zn-SOD from A.
pleuropneumoniae provides protection against N, meningitides infection. Thus _g-the monoclonal antibody is suitable for use in treating acute cases of meningococcal disease as well as in providing passive immunity to future meningococcal disease.
In a specific embodiment of the invention the antibody provides protective immunity to meningococcal disease. Thus it is suitable for use in preparations that provide passive immunity to infection and also for treatment of individuals already suffering from meningococcal infection.
In further specific embodiments of the invention the antibody provides protective immunity to bacterial infection from a range of bacterial pathogens including Actinobacillus pleuropneumoniae, Pasteurellaceae species, Neisseria species and Haemophilus species.
In a further preferred embodiment of the invention the antibody displays bactericidal activity. This activity may be directly attributed to the antibody itself or mediated via the complement system.
A fifth aspect of the invention provides for a multivalent vaccine comprising a plurality of Cu,Zn-SODs of the dimeric type, or fragments, derivatives or variants thereof, wherein said plurality of Cu,Zn-SODs are from the same or different species of Gram negative bacteria. A specific embodiment of the invention provides a multivalent vaccine comprising a plurality of Cu,Zn-SODs of the dimeric type, or fragments, derivatives or variants thereof, and one or a number of different bacterial proteins, or a fragment, derivative or variant thereof, that is not or are not a Cu,Zn-SOD. A further preferred embodiment of the invention is a multivalent vaccine that provides protective immunity to meningococcal infection.
A sixth aspect of the invention provides for use of a Cu,Zn-superoxide dismutase of the dimeric type, or a fragment, derivative or variant thereof, in the manufacture of a medicament, e.g. a vaccine, for use against bacterial infection. In a preferred embodiment of the invention the use is in response to a bacterial infection due to Gram negative species of bacteria. In a specific embodiment of the invention the use is in response to a meningococcal infection. This aspect also provides a method of treating bacterial, particularly meningococcal, infection by administering an effective amount of a Cu,Zn-SOD or a fragment, variant or derivative thereof, or other composition according to the invention.
A seventh aspect of the invention provides for an antibody specific to bacterial Cu,Zn-SOD of the dimeric type, or a fragment, derivative or variant thereof. A preferred embodiment of the invention provides for a monoclonal antibody (MAb) that is specific to bacterial Cu,Zn-SOD.
There now follow examples of specific embodiments of the invention.
Example 1 Cu Zn-SOD Monoclonal Antibody Monoclonal antibodies (MAbs) were raised in mice against a glutathione-S
transferase fusion protein with the Cu,Zn-SOD from Actinobacillus pleuropneumoniae. The whole fusion protein as expressed in E. coli was found to be enzymically active. Of 72 potential hybridomas screened by western blotting, two recognised only A, pleuropneumoniae Cu,Zn-SOD, but one also recognised Cu,Zn-SOD from H. parainfluenzae, H. ducreyi, N.
meningitides and A, actinomycetemcomitans. The latter MAb was used for the passive protection studies.
Passive Protection Three experiments were performed and all demonstrated that the MAb protects mice against lethal infections with N, meningitides. Adult NIH mice were given an intra-peritoneal tip) injection with antibody (50,u1) per mouse 2h before challenge with Neisseria meningitides. Mice were given further ip injections of antibody 2, 5 and 24h after the challenge. The N. meningitidis challenge dose was the stated number of viable bacteria (see tables of results), given as an 0.5m1 ip injection, containing 2mg iron dextran. At 24h mice were given a further 2mg iron dextran in an 0.2m1 ip dose. Mice were then observed for 72h and the health of the animals noted. The results of these experiments are shown in the following tables.
WO 00/i2718 PCT/GB99/02828 EXPERIMENT
VACCINE CHALLENGE SURVIVORS
/ CHALLENGED
DOSE
growth 105 5/5 4/5 4/5 medium growth 106 5/5 3l5 3/5 medium 50,u1 MAb 105 515 5/5 5/5 50,u1 MAb 106 5/5 5/5 5/5 5~r1 MAb 105 5/5 5/5 5/5 5NI MAb 106 5/5 4/5 4/5 Polyclonal 105 5/5 5/5 5/5 serum Polyclonal 106 5/5 5/5 5/5 1~ serum '"
- raised against meningococcal outer membrane vesicles.
EXPERIMENT
VACCINE CHALLENGE SURVIVORSJCHALLENGED
Growth 2 x 105 5/5 5l5 5!5 medium Growth 2 x 1 Os 515 1 /5 0/5 medium 50NI MAb 2 x 105 5!5 5/5 5/5 IO 50,u1 MAb 2 x 108 5/5 5/5 5/5 5,u1 MAb 2 x 105 5/5 5/5 5/5 5N1 MAb 2 x 106 5l5 3/5 3/5 Polyclonal 2 x 105 5/5 5/5 5l5 serum *' Polyclonal 2 x 106 5/5 5l5 5/5 serum ~' * - raised against meningococcal outer membrane vesicles.
WO 00/12718 PCTlGB99/02828 EXPERIMENT
VACCINE CHALLENGE SURVIVORS
/ CHALLENGED
Growth 1 x 10' 5/5 0/5 0/5 0/5 medium 50,u1 MAb 1 x 10' 5/5 4/5 3/5 2/5 5,u1 MAb 1 x 10' 5/5 1 /5 0/5 0/5 Polyclonal 1 x 10' 5/5 1 /5 0/5 0/5 serum 50N1 Yersinia1 x 10' 5/5 1 /5 0/5 0/5 MAb 5irl Yersinia1 x 10' 5/5 0/5 0/5 0/5 MAb Bactericidal Activity In addition to passive protective activity, the Cu,Zn-SOD MAb has been found to be bactericidal to two strains of meningococcus, with titres of 64-128 compared to a titre of 256 for a Group B polysaccharide specific MAb.
Example 2 Cloning and Expression of Cu,Zn-SOD - Purification of protein via a nickel affinity column Genomic DNA isolated from N.meningitidis strain MC58 was used as a template to amplify the sodCgene (Cu,Zn-SOD). Primers were designed using the information in the Genbank database. The 5' primer fSEQ ID N0.1 ) incorporates an Ndel site at the level of the ATG start codon, allowing the PCR product to be cloned into a variety of pET (Novagen) and pMTL (CAMR) vectors for expression. The 3' primer (SEQ ID N0.2) generates a polyhistidine (6 x His) tag in the translated protein to facilitate nickel affinity purification.
The primer sequences are as follows:
"SEQ ID N0.1 " : 5' primer: 5' GGC ATA TGA ATA TGA AAA CCT TAT TAG
3' "SEQ ID N0.2" : 3' primer: 5' GGG CTG AGC TTA TTA GTG GTG GTG GTG
GTG GTG TTT AAT CAC GCC ACA TGC CAT ACG TG 3' The products were sub-cloned into pET22b and transferred to BL21 DE3 for initial expression. His-tagged protein was purified on a nickel charged Hi-Trap column.
Example 3 Purification of protein via a glutathione affinity column - A glutathione-S-transferase (GST) fusion was generated by cloning the sodC
gene of Example 2 (described above) into one of the pGEX series of vectors (Pharmacia). An N-terminal fusion was generated by cloning a sodC PCR
product as an EcoRl-Xhol fragment in frame into pGEX-4T-1 (restriction sites generated by PCR primers containing these sites in-frame immediately upstream or downstream of the sodC coding sequence, including 3' stop codon). The expressed protein was a GST-SodC fusion linked by a thrombin cleavage site. The recombinant protein was purified on a glutathione affinity column and cleaved with thrombin to release SodC. This SodC protein has an additional five amino acids on the N terminus, compared to wild type protein (sequence GSPQF).
Example 4 Purification of protein via a glutathione affinity column - Alternative method This alternative cloning strategy varies from the method of Example 3 in that it allows reduction in the number of additional N terminal amino acids on the cleaved SodC product. This is achieved by removing the internal MC58 sodC
BamHl restriction site by site directed mutagenesis. PCR amplification of this altered gene with a 5' BamHl site in-frame upstream of the ATG start codon facilitates cloning of a BamHl-EcoRl fragment into pGEX-2T vector (Pharmacia). The resultant GST-SodC fusion protein is expressed and SodC
released by cleavage with thrombin protease, giving rise to SodC product with just a two amino acid N terminal 'tag' (sequence GS).
References:
Farrant, J.L. et al. (1997); Bacterial copper- and zinc- cofactored superoxide dismutase contributes to the pathogenesis of systemic saimonellosis;
s Molecular Microbiology 25(4), 785-796.
Kroll, J.S., Langford, P.R, Wilks, K.E., Keil, A.D. (1995); Bacterial Cu,Zn-superoxide dismutase: phylogenetically distinct from the eukaryotic enzyme, and not so rare after all!; Microbiology 141, 2271-2279.
io Pesce, A. et al (1997); Unique structural features of the monomeric Cu,Zn-superoxide dismutase from Escherichia coli revealed by X-ray crystallography;
J. Mol. Biol 274, 408-420.
is Tabatabai, L.B., Pugh, G.W. (1994); Modulation of immune responses in Balb/c mice vaccinated with Brucella abortus Cu,Zn-superoxide dismutase synthetic peptide vaccine; Vaccine 12( 10), 919-924.
Wilks, K.E. et al. (1998); Periplasmic superoxide dismutase in meningococcal 2o pathogenicity; Infection and Immunity, 66., 213-217.
SEQUENCE LISTING
<110> Microbiological Research Authority Imperial College School of Medicine Gorringe, Andrew Richard .
Langford, Paul Richard Kroll, John Simon Robinson, Andrew <120> SUPEROXIDE DISMUTASE AS A VACCINE ANTIGEN
<130> 20860pct camr <140>
<141>
<160> 2 <170> PatentIn Ver. 2.1 <210> 1 <211> 27 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: primer <400> 1 ggcatatgaa tatgaaaacc ttattag 27 <210> 2 <211> 59 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: primer <400> 2 gggctgagct tattagtggt ggtggtggtg gtgtttaatc acgccacatg ccatacgtg 59
Claims (27)
1. A pharmaceutical composition for vaccination, comprising:-(i) a bacterial Cu,Zn-superoxide diamutase (Cu,Zn-SOD) of the dimeric type, or a fragment, variant or derivative of the Cu,Zn-SOD, wherein antibodies raised against said fragment, variant or derivative also bind intact full length Cu,Zn-SOD; or (ii) a nucleic acid coding for the Cu,Zn-SOD fragment, variant or derivative;
and a pharmaceutically acceptable carrier.
and a pharmaceutically acceptable carrier.
2. A pharmaceutical composition according to Claim 1, wherein said composition provides protection against meningococcal infection.
3. A pharmaceutical composition according to Claim 1 or 2, wherein said composition provides protective immunity to Actinobacillus pleuropneumoniae infection.
4. A pharmaceutical composition according to Claims 1 or 2, wherein said composition provides protective immunity to infection from a gram negative bacterial species selected from the group consisting of Pasteurellaceae:
Neisseria; Haemophilus; Salmonella: and Escherichia.
Neisseria; Haemophilus; Salmonella: and Escherichia.
5. A pharmaceutical composition according to any previous claim, wherein the Cu,Zn-SOD is obtainable from a recombinant gene cloned from bacteria.
6. A vaccine comprising (i) a bacterial Cu,Zn-superoxide dismutase (Cu,Zn-SOD) of the dimeric type, or a fragment, variant or derivative of the Cu,Zn-SOD, wherein antibodies raised against said fragment, variant or derivative also bind intact full length Cu,Zn-SOD; or (ii) a nucleic acid coding for the bacterial Cu,Zn-SOD fragment, variant or derivative.
7. A vaccine according to Claim fi, wherein the Cu,Zn-SOD is obtainable from a recombinant gene cloned from bacteria.
8. A vaccine according to Claims 6 or 7, wherein said vaccine provides protection against meningococcal infection.
9. A method of preparing a pharmaceutical composition comprising:-1) isolating a gene for a bacterial Cu,Zn-SOD of the dimeric type or a fragment, variant or derivative of the Cu,Zn-SOD, wherein antibodies raised against said fragment, variant or derivative also bind the full length intact Cu,Zn-SOD; and 2) (a) synthesising the Cu,Zn-SOD fragment, variant or derivative from the gene; and combining said Cu,Zn-SOD, fragment, variant or derivative, with a pharmaceutically acceptable carrier, or (b) combining said gene with a pharmaceutically acceptable carrier.
10. A pharmaceutical preparation comprising an antibody to a bacterial Cu,Zn-SOD of the dimeric type, or a fragment, derivative or variant of the Cu,Zn-SOD, wherein antibodies raised against said fragment, derivative or variant also bind intact full length Cu,Zn-SOD; and a pharmaceutically acceptable carrier.
11. A pharmaceutical preparation according to Claim 10, wherein said antibody provides protective immunity to meningococcal disease.
12. A pharmaceutical preparation according to Claim 10, wherein said composition provides protective immunity to Actinobacillus pleuropneumoniae infection.
13. A pharmaceutical preparation according to Claim 10 or 11, wherein said composition provides protective immunity to infection from a gram negative bacterial species selected from the group consisting of Pasteurellaceae;
Neisseria; Haemophilus; Salmonella; and Escherichia.
Neisseria; Haemophilus; Salmonella; and Escherichia.
14. A pharmaceutical preparation according to any of Claims 10 to 13, wherein said antibody displays bactericidal activity.
15. A multivalent vaccine comprising a plurality of Cu,Zn-SODs of the dimeric type, or fragments, derivatives or variants thereof, wherein antibodies raised against said fragments, derivatives or variants also bind intact full length Cu,Zn-SOD, and wherein sand plurality of Cu,Zn-SODS are from the same or different species of Gram negative bacteria.
16. A multivalent vaccine comprising a bacterial Cu,Zn-SOD of the dimeric type, or fragments, derivatives or variants thereof, wherein antibodies raised against said fragments, derivatives ar variants also bind intact full length Cu,Zn-SOD; and a second protein that is not a Cu,Zn-SOD.
17. A multivalent vaccine according to Claims 15 or 16, wherein said vaccine provides protective immunity to meningococcal disease.
18. Use of a bacterial Cu,Zn-superoxide dismutase of the dimeric type, or a fragment, derivative or variant of the Cu,Zn-SOD, wherein antibodies raised against said fragment, derivative or variant also bind intact full length Cu,Zn-SOD; in the manufacture of a medicament for treatment or prevention of bacterial infection.
19. Use according to Claim 18, wherein the bacterial infection is due to Gram negative species of bacteria.
20. Use according to Claims 18 or 19 wherein the bacterial infection is due to meningococcal infection.
21. Use according to Claim 18, wherein said composition provides protective immunity to Actinobacillus pleuropneumoniae infection.
22. Use according to Claim 19, wherein said gram negative bacterial species selected from the group consisting of Pasteurellaceae; Neisseria;
Haemophilus; salmonella; and Escherichie.
Haemophilus; salmonella; and Escherichie.
23. Use of a nucleic acid encoding a bacterial Cu,Zn-superoxide dismutase of the dimeric type, or a fragment, derivative or variant of the Cu,Zn-SOD, wherein antibodies raised against said fragment, derivative or variant also bind intact full length Cu,Zn-SOD, in the manufacture of a vaccine against bacterial infection.
24. Use of an antibody specific to bacterial Cu,Zn-SOD of the dimeric type, or a fragment of the antibody, in the manufacture of a medicament for treatment or prevention of bacterial infection.
25. Use according to Claim 24 wherein the antibody is a monoclonal antibody.
26. Use according to Claims 24 or 25 wherein the bacterial infection is due to meningococcal infection.
27. A method of treating or preventing bacterial infection comprising administering art effective amount of a bacterial Cu,Zn-SOD or fragment, variant or derivative of the Cu,Zn-SOD, wherein antibodies raised against said fragment, variant or derivative also bind intact full length Cu,Zn-SOD.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GBGB9818756.0A GB9818756D0 (en) | 1998-08-27 | 1998-08-27 | Superoxide dismutase as a vaccine antigen |
| GB9818756.0 | 1998-08-27 | ||
| PCT/GB1999/002828 WO2000012718A1 (en) | 1998-08-27 | 1999-08-27 | Superoxide dismutase as a vaccine antigen |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2341639A1 true CA2341639A1 (en) | 2000-03-09 |
Family
ID=10837964
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002341639A Abandoned CA2341639A1 (en) | 1998-08-27 | 1999-08-27 | Superoxide dismutase as a vaccine antigen |
Country Status (6)
| Country | Link |
|---|---|
| EP (1) | EP1108038A1 (en) |
| JP (1) | JP2002523521A (en) |
| AU (1) | AU761394B2 (en) |
| CA (1) | CA2341639A1 (en) |
| GB (1) | GB9818756D0 (en) |
| WO (1) | WO2000012718A1 (en) |
Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005019828A1 (en) | 2003-08-20 | 2005-03-03 | Amorfix Life Sciences Ltd. | Epitope protection assay and method for detecting protein conformations |
| US7794692B2 (en) | 2005-12-02 | 2010-09-14 | Amorfix Life Sciences Ltd. | Methods and compositions for detecting amyotrophic lateral sclerosis |
| US7887803B2 (en) | 2005-12-02 | 2011-02-15 | Amorfix Life Sciences | Methods and compositions to treat misfolded-SOD1 mediated diseases |
| WO2007098607A1 (en) | 2006-03-03 | 2007-09-07 | Amorfix Life Sciences Ltd. | Methods and compositions to treat and detect misfolded-sod1 mediated diseases |
| CN108822192B (en) * | 2018-06-13 | 2020-11-06 | 华中师范大学 | Actinobacillus pleuropneumoniae immunoprotective antigen protein APJL _1976 and application thereof |
| CN108840913B (en) * | 2018-06-13 | 2020-11-06 | 华中师范大学 | Actinobacillus pleuropneumoniae immunoprotective antigen protein APJL _0922 and application thereof |
| CN108794584B (en) * | 2018-06-13 | 2021-02-19 | 华中师范大学 | Actinobacillus pleuropneumoniae immunoprotective antigen protein APJL _1380 and application thereof |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0474134A (en) * | 1990-07-13 | 1992-03-09 | Fujisawa Pharmaceut Co Ltd | Subcutaneously injecting preparation for prevention and therapy of nephrosis syndrome and nephritis |
| DE4038563A1 (en) * | 1990-12-04 | 1992-06-11 | Gruenenthal Gmbh | USE OF SUPEROXIDE DISMUTASES FOR PROPHYLAXIS AND / OR TREATMENT OF ORGAN FAILURE IN RISK PATIENTS WITH POLYTRAUMA |
| JPH04327541A (en) * | 1991-04-23 | 1992-11-17 | Nippon Kayaku Co Ltd | Agent for treating damage of implanted organ caused by rejection thereof |
| FR2675997B1 (en) * | 1991-05-03 | 1993-12-24 | Oreal | TOPICAL ANTI FREE RADICAL COMPOSITION BASED ON SUPEROXIDE DISMUTASE AND A PHOSPHONIC DERIVATIVE. |
-
1998
- 1998-08-27 GB GBGB9818756.0A patent/GB9818756D0/en not_active Ceased
-
1999
- 1999-08-27 CA CA002341639A patent/CA2341639A1/en not_active Abandoned
- 1999-08-27 JP JP2000567704A patent/JP2002523521A/en active Pending
- 1999-08-27 EP EP99943065A patent/EP1108038A1/en not_active Withdrawn
- 1999-08-27 WO PCT/GB1999/002828 patent/WO2000012718A1/en not_active Application Discontinuation
- 1999-08-27 AU AU56350/99A patent/AU761394B2/en not_active Ceased
Also Published As
| Publication number | Publication date |
|---|---|
| JP2002523521A (en) | 2002-07-30 |
| WO2000012718A1 (en) | 2000-03-09 |
| EP1108038A1 (en) | 2001-06-20 |
| AU5635099A (en) | 2000-03-21 |
| AU761394B2 (en) | 2003-06-05 |
| GB9818756D0 (en) | 1998-10-21 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| EEER | Examination request | ||
| FZDE | Discontinued |